Antibody variants

ABSTRACT

This invention relates to an antibody which is modified version of a therapeutic antibody with affinity for a cell-surface antigen, the antibody having reduced affinity for the antigen compared with the therapeutic antibody as a result of a modification or modifications to the antibody molecule, wherein the antibody is capable of inducing immunological tolerance to the therapeutic antibody. The invention further relates to a method of inducing immunological tolerance to a therapeutic antibody, comprising administering to a patient an antibody which is a modified version of the therapeutic antibody and which has reduced affinity for the antigen as compared with the therapeutic antibody.

This invention relates to modified antibodies for inducing immunologicaltolerance in human beings or animals.

Antibodies, or immunoglobulins, comprise two heavy chains linkedtogether by disulphide bonds and two light chains, each light chainbeing linked to a respective heavy chain by disulphide bonds. Each heavychain has at one end a variable domain followed by a number of constantdomains. Each light chain has a variable domain at one end and aconstant domain at its other end, the light chain variable domain beingaligned with the variable domain of the heavy chain and the light chainconstant domain being aligned with the first constant domain of theheavy chain. The constant domains in the light and heavy chains are notinvolved directly in binding the antibody to antigen.

The variable domains of each pair of light and heavy chains form theantigen binding site. The variable domains of the light and heavy chainshave the same general structure; each domain comprises four frameworkregions, whose sequences are relatively conserved, connected by threecomplementarity determining regions (CDRs). The CDRs are held in closeproximity by the framework regions. CDRs from adjacent light and heavychain variable domains together contribute to the formation of theantigen binding site.

BACKGROUND OF THE INVENTION

Antibodies directed to specifically chosen antigens have been used inthe treatment of various conditions. For example, Campath-1 monoclonalantibodies (mAb) have been used successfully to induce remissions inlymphoma and leukemia patients and for the treatment of rheumatoidarthritis and vasculitis. The target antigen, CD52 (also referred to asCDw52; see e.g. Xia et al., 1991), is a GPI-anchored glycoprotein oflymphocytes and monocytes (and parts of the male reproductive system).It has an exceptionally short peptide sequence of 12 amino acids and asingle, complex, N-linked oligosaccharide at Asn3 (Hale et al, 1990; Xiaet al, 1991). CD52 is a good target for antibody-mediated killing and istherefore an effective cell surface molecule for various therapeuticregimens in which reduction in lymphocytes is an objective (e.g. removalof cells from donor bone marrow to prevent graft-versus-host disease,treatment of leukemia and lymphoma, and immuno-suppression).

Several rat anti-human CD52 Campath-1 mAb were generated by fusion ofthe Y3 rat myeloma line with spleen cells from a rat immunized withhuman T lymphocytes (Hale et al, 1983). Although the clinicaleffectiveness of rat Campath-1 mAb has been demonstrated regularly, manypatients mounted an anti-antibody (antiglobulin) response against thexenogeneic protein that prevented retreatment with the therapeuticantibody. Antibody therapy is often limited by the antiglobulinresponse. The anti-idiotypic component (anti-Id; directed against the AbV regions and in particular the Ab-combining site) inhibits the bindingof the Ab to its target while both the anti-Id and the anti-isotypiccomponent (directed against the constant regions) act to accelerateantibody clearance. A major concern is the neutralizing effect of theantiglobulin response. As with antiglobulin responses in general,anti-Id responses interfere with the clinical potency of a therapeuticAb by forming Ab aggregates that are rapidly cleared from thecirculation, reducing the chance for interaction with target antigen.Unfortunately, most antiglobulin sera contain anti-Id antibodies. Thishas been demonstrated for a number of therapeutic mAb and is especiallynoted after repeated treatments.

To reduce the immunogenicity of the rat IgG2b Campath-1 antibody,YTH34-5, the gene fragments encoding the VL and VH were humanized by“CDR grafting” of the rodent hypervariable regions onto human frameworkregions (Jones et al, 1986; Reichmann et al, 1988). This was carried outby splicing the CDR sequences encoding the rat Campath-1 antibody ontosequence encoding human framework backbone provided by thecrystallographically solved myeloma proteins NEW (for the VH) and REI(for the VL). The resulting protein had low antigen-binding titre andmodelling of the humanized V-region showed that residue 27 in the VHframework sequence was critical for preserving the loop structure ofCDR1. This residue was changed from the residue found in NEW (Ser) backto the rat residue Phe which resulted in restoration of antigen binding.During the to modelling, an additional change (NEW residue Ser to therat residue Thr) was also suggested. However, in functional assays thissubstitution had no effect on antigen binding, but the double mutant(Ser27 to Phe27 and Ser30 to Thr30) expressed the most protein andtherefore was used to produce therapeutic humanized Campath-1 Ab,designated Campath-1H (Reichmann et al, 1988). As many human VHframeworks have threonine at position 30, this change was not consideredan additional risk to the antibody's immunogenicity. The humanized VLand VH were then genetically fused with human light chain and heavychain constant regions, respectively. In summary, the humanizedCampath-1 antibody consists of human residues at all positions exceptthose encoding the 3 CDRs of the light chain, the 3 CDRs of the heavychain, and residues Phe27 and Thr30 in VH of the heavy chain.

In clinical trials, the humanized version (Campath-1H) was found to bemuch less immunogenic than the rat IgG2b Campath-1 antibody.Humanization reduces the immunogenicity of rodent mAb, although both theidiotype and the allotype of a humanized mAb might stilt be targets forhumoral responses. Sensitization to idiotype has indeed been documentedin some allotype-matched recipients of Campath-1 H (Isaacs et al, 1992;Lockwood et al, 1993). These responses were revealed by the presence ofanti-id in the patients' sera. One patient generated high-titre anti-Idthat crossreacted on the entire panel of CD52 mAb. Humanized Campath-1antibodies are described in EP 0 328 404 the teachings of which areincorporated herein by reference, in their entirety.

One strategy to further reduce the immunogenicity of Campath-1H might beto re-graft the 6 CDR loops onto well-characterized human germlineframework regions. The majority of the humanized V regions so far haveused rearranged V-genes as acceptor framework sequence. This was thecase for Campath-1H as framework sequences from myeloma proteins wereused to provide acceptor sequences for both VH and VL. RearrangedV-genes often contain somatic mutations, acquired during the process ofaffinity maturation. These will be unique to the individual from whichthe rearranged genes were derived and therefore may be seen as foreignin another individual. However, there is a possibility that regraftingmay introduce new idiotypic epitopes, formed by the junctional regionsencompassing CDR residues and new framework residues. Furthermore,humanization alone may not solve the problem of anti-Id responsesbecause the human population is outbred and it is unlikely that allpatients will be tolerant to a given humanized mAb. Even in antibodyconstant regions, there are a number of different alleles which carryallotypic markers to which naturally occurring antiglobulin responsescan be demonstrated. The problem is more complex for V-region segments,which show a higher degree of variation both in allotype and haptotypein comparison to constant regions.

Another approach is to induce tolerance to the potentially foreignpeptides contained within the Campath-1H V-region. We know that theantiglobulin response is itself a B-cell response which is CD4+ T-celldependent. Isaacs and Waldmann (1994) demonstrated that mice deprived ofCD4+ Tells were unable to respond to a foreign cell-binding mAb (ratanti-mouse CD8 mAb). CD4+ Tell depletion was carried out by adultthymectomy combined with administration of a depleting CD4 mAb. In thesemice, the response to subsequently administered mAb or SRBC wasmeasured. CD4+ T-cell deficient mice failed to make either anantiglobulin response or an anti-SRBC response, demonstrating that theanti-Ig response, like the anti-SRBC response, is classically CD4+T-cell dependent. In order to generate T-cell help and to get theappropriate T-cell response, the adminstered Ab must be processed as aprotein antigen and presented, presumably in the context of an MHC classII molecule, by a suitable antigen presenting cell. Therefore, two mainstrategies can be adopted to decrease the immunogenicity of a humanizedV-region. (1) We can “silence” the antibody molecule itself, adoptingstrategies to eliminate any potential T helper epitopes, or (2) we canpresent all the potential T helper epitopes in a manner that inducestolerance instead of reactivity to those epitopes.

“Silencing” the Antibody Molecule:

a) In theory, we might be able to silence the antibody itself so thatthe immune system will not recognize foreign determinants. This would bepossible if we could scan the VL and VH amino acid sequences for motifsthat could bind to MHC class II molecules. If we could thus identify keyresidue(s) in a potential class II peptide that were not involved inantibody specificity or affinity, then it/they could be changed bysite-directed mutagenesis to residue(s) that did not allow associationwith class II molecules. T helper peptides are not random, and anyprotein has only a limited number of peptides capable of binding to MHCclass II molecules, and also to T-cell antigen receptors. However, thisis not possible at present because class II-binding peptides are not yetcharacterized to a sufficient degree to be identified by scanningprotein sequences. This is in part due to the heterogeneous nature ofclass II peptides. Naturally processed peptides isolated from MHC classII molecules are generally larger in size, variable in length and haveboth ragged ends at C- and N-termini in comparison to processed peptidesisolated from MHC class I molecules. Whereas class derived peptides aremostly of uniform length of 8-9 amino acid residues, MHC classII-associated peptides range from 12-24 amino acids (Rudensky et al,1991; Hunt et al, 1992; Rudensky and Janeway, 1993). Class I-derivedpeptides have sequence motifs with specific anchor residues in certainpositions allowing their side chains to fit in the binding pockets ofthe peptide-binding groove, and the peptide-binding groove is closed atboth ends. In contrast, class II peptides are bound in an extendedconformation that projects from both ends of an “open-ended”antigen-binding groove; a prominent non-polar pocket into to which ananchoring peptide side chain fits near one end of the binding groove(Brown et al, 1993).

b) Other strategies that might be adopted to “silence” the antibody ifwe could predict class II peptide-binding motifs. For example, one couldinclude insertion of a protease cleavage site within any potential classII epitope to increase the chance of peptide degredation before theycould be presented in the context of class II. Alternatively, insertionof motifs into a V-region such as Gly-Ala repeats may inhibit thedegradation of the V-region into peptides that could associate withclass II molecules. In one system, it was shown that EBNA1 Gly-Alarepeats generated a cis-acting inhibitory signal that interfered withantigen processing during MHC class I-restricted presentation such thatCTL recognition was inhibited (Levitskaya et al, 1995). Although eitherof these approaches may hold some promise in the future, they again relyon prediction of potential MHC class II peptides from protein sequenceof humanized VL and VH regions and are therefore limited by insufficientknowledge regarding consensus motifs for class II peptides.

Inducing Tolerance to T Helper Epitopes

In lieu of sufficient knowledge regarding class II peptide motifs, wehave turned our attention toward induction of tolerance to therapeuticantibodies. In 1986, Benjamin et al and Cobbold et al described anunexpected property of cell-binding mAb: whereas it was possible toinduce tolerance to the Fc region (anti-isotype tolerance), the idiotyperemained antigenic under equivalent conditions. Moreover, it wasrelatively easy to induce tolerance to non-cell binding mAb butcell-binding mAb were found to be very immunogenic.

Isaacs and Waldmann (1994) in a preliminary study used anon-cell-binding “mixed molecule” derivatives of a cell-binding Ab toinduce tolerance to the wild-type form. The cell-binding antibody was ananti-CD8 mAb in a mouse model. The non-cell-binding derivatives weremade by pairing the relevant L- and H-chains with an irrelevant H- orL-chain, respectively. The relevant H-chain paired with an irrelevantL-chain was obtained by limiting dilution cloning of the originalhybridoma that was expressing a myeloma light chain (from the Y3 fusionpartner), as well as the specific anti-CD8 H- and L-chains. A variant ofthe hybridoma that expressed the myeloma L-chain and the specificanti-CD8 H-chain but no anti-CD8 L-chain was obtained. A cloneexpressing the relevant L-chain only was also obtained in this manner.That clone was then fused to a hybridoma expressing an irrelevantspecificity (anti-human CD3) and a variant was selected that expressedthe relevant anti-CD8 L-chain with the irrelevant anti-CD3 H-chain.Because proteins are processed into peptides prior to presentation toT-cells, helper peptides from antigen-specific H- and L-chains would be“seen” by T-cells, regardless of their partner chain. However, in thiscase, there was no advantage in tolerance induction usingnon-cell-binding mixed molecule derivatives of a therapeutic mAb in vivocompared to an isotype-matched control, suggesting that in the strain ofmice used, most (or all) of the helper epitopes were located within theconstant region.

In practice, using these “mixed molecules” of antigen-specific andirrelevant immunoglobulin chains for human therapy would not be feasiblebecause the irrelevant H- and L-chains would carry some helper epitopesthemselves, thus complicating the ability to achieve tolerance to therelevant H- and L-chains. Nor would one expect to tolerize those B-cellswhich “see”. idiotypic determinants formed by the combination of therelevant H- and L-chains of the antibody.

Campath-1 is a cell-binding mAb, and an effective tolerogen for use withit, such as a non-cell-binding form of the therapeutic mAb wouldtherefore be advantageous. The same goes for other therapeuticantibodies which have cell-binding properties, and non-cell-bindingvariants thereof.

The Invention

The invention therefore provides an antibody which is a modified versionof a therapeutic antibody with affinity for a cell-surface antigen, saidantibody having reduced affinity for the antigen compared with thetherapeutic antibody as a result of a modification or modifications tothe antibody molecule, wherein the antibody is capable of inducingimmunological tolerance to the therapeutic antibody.

Preferably, the affinity of the antibody according to the invention forthe antigen is reduced to 50% or less of the affinity of the therapeuticantibody for the antigen. More preferably, the affinity is reduced to10% or less, or to 1% or less of the affinity of the therapeuticantibody. The affinity needs to be sufficiently reduced to allow theantibody according to the invention to act as a tolerogen with respectto the therapeutic antibody. The term “non-cell-binding variant” is usedherein to refer to antibodies according to the invention, althoughantibodies according to the invention may still have some bindingaffinity for the cell surface antigen.

The ability of the antibody according to the invention to induceimmunological tolerance to a therapeutic cell-binding antibody relies onthe presence in the non-cell-binding antibody of at least one epitopealso present in the therapeutic antibody, which induces an immuneresponse in the intended patient.

The non-cell-binding antibody is preferably capable of tolerising toanti-idiotypic responses, at least to the V domain hypervariable regionsof the therapeutic antibody and preferably also to the frameworkregions. Thus it is desirable that the tolerising antibody has an aminoacid sequence similar to the therapeutic antibody in those regions.Preferably there is >90%, or >95% or >99% amino acid sequence identitybetween the variable domains of the non-cell-binding antibody and thetherapeutic antibody. Most preferably the differences are restricted toany amino acid to substitution(s) required to sufficiently reduceantigen binding affinity in the non-cell-binding antibody.

Preferably also the non-cell-binding antibody is capable of inducingtolerance to the constant regions of the therapeutic antibody. Thus, itis preferred that the constant domains of the non-cell-binding antibodyare similar to those of the therapeutic antibody, having forexample >90% or >95% or >99% amino acid sequence identity. Mostpreferably, the constant domains of the non-cell-binding antibody andthe therapeutic antibody are identical and are thus matchedallotypically.

The invention further provides fragments of an antibody describedherein, the fragments having tolerance-inducing capability. Suchfragments include monovalent and divalent fragments such as Fab, Fab′and F(ab′)₂ fragments. Also included are single chain antibodies. Thepreferred features of such fragments are as described herein in relationto non-cell-binding antibodies according to the invention. Thenon-cell-binding fragments may be for use with corresponding therapeuticantibody fragments, or with therapeutic antibody molecules.

The reduced binding affinity of the non-cell-binding antibodies may beachieved in a variety of ways. In the preferred embodiment describedherein, an alteration in the CDRs comprising one or two or more aminoacid substitutions reduces binding affinity, Alternatively, amino acidsubstitutions in other parts of the antibody molecule may be used toreduce binding affinity. For example, amino acid substitutions in theframework regions are known to significantly affect binding affinity(Reichmann et al 1988). Another alternative is a monovalent form of thetherapeutic antibody. Monovalent antibodies have reduced bindingaffinity compared to their bivalent counterparts. Monovalent forms maybe for example Fab fragments, or single chain antibodies, or any othergenetically engineered antibody fragments retaining a single bindingsite. Monovalent variants can also be produced by mutating the cysteineresidue which participates in interchain (H-H) disulphide formation(e.g., cys→ser or cys→ala). The reduction in binding affinity of amonovalent antibody compared to its bivalent counterpart may besufficient to enable tolerance induction. Preferably, the monovalentantibody is either incapable of binding Fc receptors, or incapable ofbinding complement component C1q, or both. Either or both of theseproperties can be introduced by suitable mutations (see e.g., Morgan etal., WO 94/29351, published 22 Dec. 1994 and Winter et al., EP 0 307 434B1).

The non-cell-binding antibodies or fragments according to the inventionmay thus be one of a variety of types of antibodies or fragments,including genetically engineered antibodies or antibody fragments. Inaddition, the antibodies or fragments will generally be from a mixtureof origins. For example, they may be chimeric e.g. human constantregions with rat variable domains; or humanised or CDR grafted orotherwise reshaped (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567;Cabilly et al., European Patent No. 0 125 023 B1; Boss et al., U.S. Pat.No. 4,816,397; Boss et al., European Patent No. 0 120 694 B1; Neuberger,M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No.0 194 276 1; Winter, U.S. Pat. No. 5 225 539; Winter, European PatentNo. 0 239 400 B1; Queen et al., U.S. Pat. No. 5,585,089; Queen et al.,European Patent No. 0 451 216 B1; Adair et al., WO 91/09967, published11 Jul. 1991; Adair et al., European Patent No. 0 460 167 B1; andPadlan, E. A. et al., European Patent No. 0 519 596 A1. See also,Newman, R. et al., Biotechnology, 10: 1455-1460 (1992), regardingprimatized antibody, and Huston et al., U.S. Pat. No. 5,091,513; Hustonet al, U.S. Pat. No. 5,132,405; Ladner et al., U.S. Pat. No. 4,946,778and Bird, R. E. et al., Science, 242: 423-426 (1988) regarding singlechain antibodies). Campath-1H is considered humanised although itcontains two amino acid substitutions in the framework regions.

Ideally, the antibody according to the invention is as close as possibleto the therapeutic antibody on which it is based. Administration of sucha “minimal mutant” prior to injection of the cell-binding therapeuticmAb can be used to tolerise to all T- and most B-cell epitopes in thetherapeutic mAb. Classic experiments indicate that tolerance ismaintained more effectively by T-cells than by B-cells. But since mostB-cell responses including the anti-id response require T-cell help,even if a B-cell is responsive to a given antigen, antibody productionwill be determined by the state of responsiveness of the T-cells(Chiller et al, 1971). Thus, it will be preferable to use anon-cell-binding variant which contains the minimum differences neededto reduce its affinity for the cell-surface antigen sufficiently toenable it to be used as a tolerogen. By using techniques such as X-raycrystallography, computer modeling and site-directed mutagenesis, andalso genetic methods such as phage display, it will be possible todesign suitable non-cell-binding variants for any cell-bindingtherapeutic antibody.

The antibody according to the invention is preferably in biologicallypure form, desirably being at least 95% (by weight) free of otherbiological materials.

As used herein, the term “cell-surface antigen” means an antigen whichis found on, cell surfaces, but not necessarily exclusively on cellsurfaces.

The term “therapeutic antibody” is used herein to refer to an antibodywhich may be administered to humans or animals to have a desired effectin particular in the treatment of disease. Such therapeutic antibodieswill generally be monoclonal antibodies and will generally have beengenetically engineered.

In another aspect the invention comprises a composition foradministration to a patient comprising an antibody as described herein,together with a physiologically acceptable diluent or carrier.

In a further aspect, the invention provides a host cell or cell linewhich expresses an antibody as herein described and use of such a hostcell or cell line for the production of such an antibody.

Additional aspects of the invention include the use of an antibody asdescribed herein in the manufacture of medicament for the induction oftolerance, in particular tolerance to a therapeutic antibody.

In attached figures:

FIG. 1 shows the Campath-1H heavy chain minimal mutant constructsprepared as described in the Examples.

FIG. 2 shows the PCR mutagenesis strategy for preparing the mutantconstructs of FIG. 1.

FIG. 3 shows pGEM9zf containing wild type Campath-1H heavy chain, andsubstitution of mutant fragments in the heavy chain.

FIG. 4 shows a schematic representation of one embodiment of amonovalent non-cell-binding therapeutic antibody.

Using Rational Design to Create a “Minimal Mutant”

In one embodiment for producing a non-cell-binding variant of atherapeutic mAb, amino acid residues which are involved in binding totarget antigen are identified. Relative to the number of residues thatcomprise the VL and VH domains, those that are directly involved ininteractions with antigen are small in number (Novotny et al, 1983). Andalthough the Ab-combining site is made up of 6 hypervariable loops, 1 or2 of those loops may dominate in that interaction. If a key residue orresidues can be identified, it/they can be changed by site-directedmutagenesis to a residue that will reduce (reduce or abolish)antigen-binding. Because these residues will most likely be found withinthe hypervariable loop structures and not in the framework sequencesupporting those loops, small changes may not significantly disrupt theoverall structure of the Ab.

Model Building of Ag-binding Sites to Define Key Residues forMutagenesis:

Because the constant regions and variable regions of Ab molecules arevery similar in sequences and structures, general principles regardingAb structure have been defined using relatively few solved crystalstructures. To date, approximately 50 structures of Ab fragments havebeen included in the Brookhaven Protein Data Bank, and of these, 20%have been refined to a resolution of 2.0 angstroms or better. As thestructural knowledge base increases, comparative Ab modelling (modellingby homology) becomes more reliable since there is a greater choice ofstructural templates. Variable regions (VL and VH) of different Abstructures can be combined as a structural template after superimposingtheir most conserved residues. Side chain conformations of buriedresidues are then modelled. The CDR loops are modelled by identificationof structurally similar loop templates (often loops with the same lengthand similar sequence have similar backbone conformations). These CDRsequences often fall into canonical loop motifs (excluding H3, between50 and 95% of murine VL (kappa) and VH have loop sequences consistentwith classified canonical motifs). Canonical loop backbones can then bespliced onto the model of the framework and CDR side-chain conformationscan be modelled based on conformations of residues found atcorresponding positions in other loops of the same canonical structure.Finally the model is often refined using computer programs that minimizetroublesome stereochemical constraints.

Comparative model building is becoming widely used as the size of thestructural database increases, providing a greater range of structuraltemplates. Also, the greater range of computer programs availableensures that models are becoming increasingly accurate. For example, thesolved crystal structure of Campath-1H was very close to the structurepredicted by molecular modelling. We could predict from the to modellingdata that mutations 1 and 2 described in the Examples were likely tohave a detrimental effect on binding to CD52. The crystal structureconfirmed these predictions and also predicted that mutation 3 coulddisrupt binding to CD52.

Obviously, to create a non-cell-binding version of a therapeutic Ab, itis desirable to start with a solved crystal structure, preferablyco-crystallized with antigen so that the key contact residue(s) can beidentified and substituted for residue(s) that destroy antigen binding.However, in many cases, a good molecular model could provide thenecessary information. In cases where the molecular model is of poorquality (for example, if the appropriate structural templates do notexist in the databank), CDR swapping experiments (as described in theExamples) will provide information on which CDRs must be targeted formutation. Alanine scanning mutagenesis (mutating each residuesequentially to Ala) through those regions could identify the keyresidue(s) involved in antigen-binding (Cunningham and Wells, 1989). Ifchanging a single residue to Ala reduced but did not destroy binding,that position could be targetted for more drastic mutations (forexample, a substitution that created in a charge difference) to furtherreduce binding, if desired.

Alternative methods for obtaining non-cell-binding versions oftherapeutic antibodies include genetic techniques such as phage displayusing error prone PCR (Gram et al, 1992) and cycling of V-region genes(e.g. as sFv constructs) through a bacterial mutator strain (e.g. mutD5)(Low et al., 1996). Such genetic methods can provide powerful screeningsystems.

The invention will now be further described in the examples whichfollow. Although the specific example of Campath-1H is given in thisdocument. the invention is not limited to antibodies based onCampath-1H. It is anticipated that other cell-binding therapeuticantibodies, especially those which would be given in repeated doses,will become more widely to accepted using this strategy.

EXAMPLES

A. Creating a “Minimal Mutant”

We have devised a method to determine which of the CDR loops of thehumanized Campath-1 mAb are the most important ones for binding to CD52.Mutant VL or VH were genetically constructed in which each of the 6hypervariable regions (as defined by Kabat et al (1987) using amino acidsequence alignments of V-regions in the protein databases) wasindividually swapped for the corresponding CDR from the V-region thathad provided the human VL or VH acceptor sequence during humanization(REI and NEW, respectively). The engineered V-regions were expressed asFab fragments in E coli using the pHEN vector (Hoogenboom et al, 1991).In this system, the pelb leader sequence was used to direct proteinexpression to the periplasm, where association of L-chain and truncatedH-chain occurs (Hoogenboom et al, 1991). When these Fab fragments wereassayed for binding to immobilized CD52, it was found that swapping the(VH) CDR2 of NEW into the humanized Campath-1 Fab completely destroyedbinding to CD52. Replacing (VH) CDR3 reduced binding to CD52 8-foldwhile replacing (VH) CDR1 and (VL) COR3 reduced binding 3-fold. Nochange in binding was detected when (VL) CDR1 or (VL) CDR2 werereplaced. From these results, it appeared that (VH) CDR2 contained a keyresidue(s) necessary for antigen-binding. DNA encoding the “wild-type”humanized Campath-1H-chain (Reichmann et al. 1988) was used as PCRtemplate for site-directed mutagenesis. This heavy chain sequenceencodes human protein at all positions except the three VH CDR regionsand positions 27 and 30 of the first framework region. We focussed onthe H2 loop within VH CDR2 to make mutations which would abolish bindingof the Ab to CD52. H2 is the actual loop structure (Chothia and Lesk,1987) that is found within the 19 amino acid VH CDR2 denoted“hypervariable” by Kabat et al's definition (Kabat et al, 1987) (seeFIG. 1). It is known that a few key residues in the loop and/orframework regions determine relatively few CDR loop conformations andcanonical loop motifs have been identified for most CDR including VHCDR2 (Chothia and Lesk, 1987). Since it is the loop structures thatstick out from the V-region β-barrel framework to make contact withantigen, mutations in the loop would have the greatest chance ofdestroying antigen binding whilst preserving Ab structure. In general,we restricted the changes to the H2 loop except for H-chain mut6 whichcontained an additional mutation in the residue immediately preceedingH2 as discussed in more detail below.

Summary of Campath-1H Heavy Chain Minimal Mutant Constructs (FIG. 1)

Mutation 1 is a single charge difference at residue 52b from Lys to Asp.It was predicted from the molecular modelling of Campath-1H Ab, andsupported by the crystal structure, that the side chain of this residueis pointing out of the Ag binding pocket, towards the approach ofantigen. Since the positive charge of the Lys is thought to interactwith the negatively charged phosphate groups of the GPI anchor of CD52,it is possible that this single mutation will destroy antigen-binding.

Mutation 2 is a single charge difference at residue 52a from Asp to Lys.It was predicted from the molecular modelling of Campath-1H Ab that thischange could interfere with antigen-binding.

Mutation 3 is a single charge difference at residue 53 from Lys to Asp.From the crystal structure of Campath-1H Ab, it is clear that themajority of this residue side chain is solvent accessible and thereforemay be involved in the interaction with the negatively charged phosphategroups of the GPI anchor of CD52, as for mutation 1.

Mutation 4 is a double mutation encompassing the individualsubstitutions of mutant 1 and 3 (Lys52b and Lys53 to Asp).

Mutation 5 is a triple mutation encompassing the individualsubstitutions of mutant 1, 2 and 3 (three charge differences: Asp52a toLys; Lys52b and Lys53 to Asp).

Mutation 6 is a triple mutation encompassing the individualsubstitutions of mutant 1 and 2 (two charge differences Asp52a to Lys;Lys52b to Asp), and an additional mutation of Arg52 to Ala. Residue 52has been shown to differ between 3 different Campath-1 Ab of high, lowand moderate affinity and may be therefore directly involved in affinitymaturation. This in turn might suggest a role in antigen binding.

For each of these heavy chain mutations, the change(s) was/were encodedon oligonucleotide primers 1B and 2A (FIG. 2). PCR was carried out on“wild-type” Campath-1 heavy chain DNA using a 5′ primer annealing to theleader sequence and containing an upstream HindIII site (primer 1A) andprimer 1B to generate a 200 bp fragment. Similarly, primer 2B (annealingto CH1 and containing a BstXI site followed by an EcoRI site) and primer2A, a 440 bp fragment was generated. These fragments were gel purifiedand then combined in a single PCR reaction. Primer 1A and primer 2B wereadded after the first cycle (thus allowing the 2 pieces of overlappingDNA to anneal before amplification). Following PCR, the fragments weregel purified and digested with HindIII and EcoRI and were transferredinto intermediate sequencing vectors (PUC19 or pGEM3zf) for verificationof sequence. A unique PstI site located in the Campath-1H VH and aunique BstXI site in the CH1 region allowed the mutant V-regions (andpartial CH1 sequence) to be isolated as PstI-BstXI fragments such thatthe mutation(s) were encoded in six different DNA cassettes flanked byPstI and BstXI sites. To create the mutant Campath-1H heavy chains, theoriginal heavy chain construct (in intermediate vector pGEM9zf) was cutwith PstI and BstXI and the fragment was removed. The remaining DNA(encoding the Campath-1H heavy chain leader sequence and the V-regionupsteam of the PstI site, plus the CH1 region downstream of the BstXIsite followed by the hinge, CH2, CH3 in pGEM9zf) was gel purified (FIG.3). Ligations were then set up in which each of the DNA cassettescontaining the mutation(s) described above was joined to the gelpurified pGEM9zf(PstI-BstXI cut Campath-1H heavy chain DNA. These sixmutagenized Campath-1H heavy chains were then isolated by digestion withHindIII and gel purification, followed by ligation into HindIII cutmammalian expression vector pBAN-2. This vector is derived from thepNH316 vector that contains a neomycin selectable marker under thecontrol of the mouse metallothionein promoter and the strong humanβ-actin promoter/polyadenylation signals for expression of the desiredgene product (Page and Sydenham, 1991). As these fragments wereintroduced into a single HindIII restriction site, orientation of eachfragment was checked by DNA sequencing.

Campath-1H Light Chain Construct for Co-transfection:

DNA encoding the humanized “wild type” Campath-1H light chain (humansequence at all residues except the three CDR in the V-region) wasisolated from an intermediate vector as a HindIII to EcoRI fragment.This fragment was gel purified and then ligated into HindIII-EcoRI cutmammalian expression vector pRDN-1. This vector is derived from the pLD9vector that contains a “crippled” dihydrofolate reductase (dhfr)selectable marker (the enhancer element of the SV40 promoter has beenremoved to allow for increased levels of gene expression in the presenceof methotrexate) and the strong human β-actin promoter/polyadenylationsignals for expression of the desired gene product (Page and Sydenham,1991).

Co-transfection of Campath-1H Light Chain DNA and Mutant Heavy ChainDNA:

The expression system used to produce high levels of humanizedCampath-1H Ab in the past is the widely used mammalian expression systemfeaturing gene amplification by the use of dihydrofolate reductase(dhfr) deficient Chinese hamster ovary (CHO) cells and the use of strongβ-actin promoters for selection and amplification of the desired geneproducts (Page and Sydenham, 1991).

The following transfections (TF) were carried out;

TF1: mock (“empty” pRDN-1 plus “empty” pBAN-2)

TF2: Light chain only (Light-chain/pRDN-1 plus “empty” pBAN-2)

TF3: Light chain/pRDN-1 plus H chain mutant 1/pBAN-2

TF4: Light chain/pRDN-1 plus H chain mutant 2/pBAN-2

TF5: Light chain/pRDN-1 plus H chain mutant 3/pBAN-2

TF6: Light chain/pRDN-1 plus H chain mutant 4/pBAN-2

TF7: Light chain/pRDN-1 plus H chain mutant 5/pBAN-2

TF8: Light chain/pRDN-1 plus H chain mutant 6/pBAN-2

TF9: Light chain/pRDN-1 plus H chain “wild-type”/pBAN-2

DNA (20 μg of light chain/pRDN-1 plus 20 μg of heavy chain/pBAN-2) wasmixed in a sterile eppendorf, ethanol precipitated, and rinsed twicewith 70% ethanol. DNA pellets were resuspended in sterile Tris-EDTA.

For each transfection, the DNA was diluted with 60 μl of 20 mM HEPES (pH7.4) in a 5 ml polystyrene tube. In another tube, 120 μl of DOTAPliposomal transfection reagent (Boehringer Mannheim) was diluted with 80μl of 20 mM HEPES (pH 7.4). Then the DNA/HEPES was added to the dilutedDOTAP, mixed gently, and left at room temperature for 15 min. Culturemedium (IMDM+5% FCS+HT) was aspirated from a T75 flask containing dhfrdeficient CHO cells growing at approximately 50% confluency. TheDNA/DOTAP was then added to the flask along with 10 ml fresh culturemedium. The flask was cultured for 24 h at 37° C. in 5% CO₂. TheDNA/DOTAP was then aspirated from the flask and the cells were given 15ml fresh culture medium. After a further 24 h, selection was initiatedby removing the culture medium and adding selection medium (IMDM+5%dialysed FCS+1 mg/ml G418). The cells were cultured at 37° C. in 5% CO₂and fresh selection medium was added as necessary. Culture supernatantswere then tested by ELISA for the presence of antibody as describedbelow.

Detection of Secreted Ab in Transfection Supernatants by ELISA:

Microtitre plates were coated with 50 μL/well anti-human Ig Fc (Sigma,catalogue number I-2136) in PBS at 2.5 μg/ml overnight at 4° C. Thecoating Ab was removed and the plates were blocked by addition of 100μl/well blocking buffer (PBS+1% BSA+5% FCS+1% heat-inactivated normalrabbit serum (NRS) overnight at 4° C. The transfection supernatants wereadded (50 μl/well) for at least 1 h at room temperature. The wells werewashed with PBS/0.5% Tween-20 (PBS/Tween). Biotinylated sheep anti-humanIg (Amersham, catalogue number RPN 1003) diluted 1/5000 in blockingbuffer or biotinylated goat anti-human kappa light chain (Sigma,catalogue number B-1393) diluted 1/1000 in blocking buffer was added (50μl/well) for 1 h at room temperature. The wells were washed withPBS/Tween and 50 μl/well ExtrAvidin-peroxidase (Sigma, catalogue numberE-2886) was added for 30 min at room temperature. The wells were washedonce more and 100 μl/well of substrate o-phenylenediaminedihydrochloride (Sigma, catalogue number P-7288) was added. Colourchange was measured at 492 nM using a Multiskan Plus microtitre platereader.

Detection of Binding to Campath-1 Antigen by ELISA:

Microtitre plates were coated with 50 μl/well-anti-mouse Ig Fc (Sigma,catalogue number M-4280) in PBS at 2.5 μg/ml overnight at 4° C. Thecoating Ab was removed and the plates were blocked by addition of 100μl/well blocking buffer (PBS+1% BSA+5% FCS+1% heat-inactivated NRS)overnight at 4° C. Purified recombinant Campath-1 Ag-fusion protein(sequence encoding the CD52 peptide backbone fused to sequence encodingmouse CH2 and-CH3 domains, and purified on a protein A column) was thenadded at 4 μg/ml to each well (50 μl/well) in PBS overnight at 4° C. Thewells were then washed with PBS/Tween and the transfection supernatantswere added (50 μl/well) for at least 1 h at room temperature. The wellswere washed with PBS/Tween and biotinylated sheep anti-human Ig(Amersham, catalogue number RPN 1003) diluted 1/5000 in blocking bufferwas added (50 μl/well) for 1 h at room temperature. The wells werewashed with PBS/Tween and 50 μl/well ExtrAvidin-peroxidase (Sigma,catalogue number E-2886) was added for 30 min at room temperature. Thewells were washed once more and 100 μl/well of substrateo-phenylenediamine dihydrochloride (Sigma, catalogue number P-7288) wasadded. Colour change was measured at 492 nM.

Assessment of Non-binding Mutants by ELISA:

“Wild-type” purified Campath-1H Ab binds strongly to recombinantCampath-1 Ag-fusion protein in ELISA assays. To provide a comparison forassessing non-cell-binding antibodies, the wild-type Ab can be titrateddown in concentration until binding is just detectable. This may bereferred to as “1 Ab binding unit”. A suitable non-binding mutant of thewild-type will not show detectable binding at many times thisconcentration e.g. 100 times, or 1000 times, or preferably 10,000 timesthis concentration of wild-type Campath-1H Ab.

Assessment of Non-binding Mutants In Vivo:

An alternative method which can be applied to assess non-bindingpotential is described. Because we know that the wild-type Campath-1H Abelicits a strong anti-immunoglobulin response in transgenic miceexpressing human CD52 (see next section for details on these mice),purified deaggregated preparations of the mutants can be used in vivo toassess whether they are immunogenic. If an anti-globulin response cannotbe detected at doses between 1 μg and 1 mg deaggregated mutant permouse, this is a good indication that the mutant is unable to bind CD52.

B. In Vivo Models of Tolerance Induction:

To test the ability of the minimal mutants of Campath-1H to tolerize tothe wild-type Campath-1H Ab in vivo, transgenic mice are used. Forexample, transgenic mice expressing human CD52 behind a murine CD2promoter to mimic the expression of CD52 on T-cells can be used.

To create such mice, a 2.8 kb genomic fragment containing the 2 exons ofthe human CD52 gene as well as 4.5 kb upstream and 3′ flanking sequenceof the human CD2 gene can be introduced into the genome of transgenicmice. It is thought that strong control regions are present 3′ to thehuman CD2 gene that determine the high levels and tissue-specificexpression of the gene (Greaves et al, 1989). By this method, fourCD52/CBA founders were established that transmitted the transgene.Indeed, when peripheral blood staining of their offspring was analysedby fluorescence activated cell sorting and 2-colour staining, it wasshown that the cells expressing mouse CD3 (T-cells) also expressed humanCD52 (D. Kioussis, unpublished data). These mice were bred tohomozygosity and greater than 95% of their T-cells express high levelsof human CD52 on the cell surface.

These mice produce a vigorous anti-globulin response (titre of 1/1000 orgreater) to wild-type Campath-1H at doses of 1 to 10 mg/mouse. Thisanti-globulin response includes an anti-id component as the CDR loopsare rat sequence. The effectiveness of the minimal mutants to tolerizeto subsequent challenge of wild-type Campath-1H Ab may be assessed inthe following ways:

1. Intravenous administration of a single dose (0.5 to 1 mg/mouse day 0)of each non-cell-binding mutant or irrelevant control Ab (deaggregatedby ultracrentrifugation) followed by challenge with 1 to 10 mg wild-typeCampath-1H Ab at 4 to 6 wks. Tail bleeds 10 days post challenge aretested by ELISA for Campath-1H anti-Id specificity.

2. Intravenous administration of multiple doses (0.5 to 1 mg/mouse) ofdeaggregated non-cell-binding mutant or control Ab over 2 months priorto challenge with 1 to 10 mg wild-type Campath-1H Ab. Tail bleeds 10days post challenge are tested by ELISA for Campath-1H anti-idspecificity. In both cases, the irrelevant control Ab is be anisotype-matched non-cell-binding Ab in mice such as Campath-9 which is ahumanized anti-CD4 Ab (Gorman et al, 1991).

C. How the Strategy Could Be Adopted For Human Therapy:

An amount (e.g. 500 mg) of the non-cell-binding form of the therapeuticantibody would be administered to a patient awaiting treatment with thetherapeutic antibody. Preferably the non-cell-binding antibody asadministered is freshly deaggregated (for example by passage through afine filter). A period of time later (for example 7 days), during whichtime the T-cells and B-cells would become tolerised, the wild-type formof the therapeutic antibody would be given.

1:). Additional considerations:

1. It should be easier to tolerize to a minimal mutant than to HGG or tomixed chain Ab molecules.

In the tolerance models of Benjamin et al (1986), tolerance topolyclonal HGG was induced in mice following depletion of CD4+ T-cells,but also using deaggregated material. It was found that tolerance tothese soluble proteins could be achieved relatively easily. Also, in thework of Isaacs and Waldmann (1994), CD4 Ab were given during toleranceinduction to the non-cell-binding mixed chain Ab molecules (irrelevantand antigen specific H- and L-chains), or non-cell-binding forms wereused as tolerogens in their own right following their deaggregation.

In our modified approach to inducing tolerance using a minimal mutant,the foreigness of the protein will be less than that of polyclonal HGGor of the mixed chain Ab molecules in mice. In cases where thetherapeutic mAb is humanized, only the CDR loops (and in some cases,some framework positions) are comprised of rodent sequence. It thereforemay be possible to tolerize with a deaggregated minimal mutant in theabsence of CD4 mAb. However, even if CD4 administration was required, ahumanized therapeutic CD4 is available (CAMPATH-9; Gorman et al, 1991).The studies in transgenic animals should address these details.

2. Creation of a monovalent form of the minimal mutant (FIG. 4).

Thus far we have considered tolerance induction using a minimal mutantthat is essentially like the wild-type therapeutic except for minimalresidue change(s) that will disrupt antigen binding. We also propose amonovalent form that is also significantly smaller than the minimalmutant.

In one embodiment, the monovalent form is a single-chain Fv [formed bythe VL, a short peptide linker (such as those reviewed in Huston et al,1991) and the mutated VH] genetically fused with the sequence encodingthe hinge-CH2-CH3 of human IgG1. This construct is expressed inassociation with a truncated heavy chain (hinge-CH2-CH3 only, Routledgeet al, 1991) such that a protein is expressed that is composedessentially of a single Ab-combining site and a functional Ig Fc domain.The immunogenicity of the different peptide linkers is expected to benegligible given their small size (generally 14 to 18 residues inlength) and abundance of small residues (eg Gly and Ser) making up thelinkers. A popular choice is the 15-residue linker (Gly₄Ser)₃ in whichthe serine residues confer extra hydrophilicity on the peptide backbone(to inhibit its intercalation between the variable domains duringfolding) and which is otherwise free of side chains that mightcomplicate domain folding (Huston et al, 1988).

SFv have been expressed in mammalian cells from a number of differentantibodies and have been shown to fold into the correct conformation forantigen-binding by functional activity (Gilliland et al, 1996). The Fcportion is a preferred feature which should ensure serum half-lifecomparable to the minimal mutant and to the wild-type therapeutic Ab,whilst monovalency will ensure that binding to CD52 is greatly reduceddue to the decrease in avidity. We have already shown from theCDR-swapping experiments (section A1) that the Campath-1 Ab binds poorlyto CD52 in a monovalent form. In addition to reducing the avidity of themolecule, the smaller size may be a bonus: in classical toleranceexperiments, it was found that the smaller the molecule, the better itwas at inducing tolerance (Parish and Ada, 1969; Anderson, 1969; Mirandaet al, 1973). By combining monovalency with a non-cell-binding mutant, ahighly effective tolerogen may be obtained.

REFERENCES

-   Anderson B. 1969. Induction of immunity and immunologic paralysis in    mice against polyvinyl pyrrolidone. J Immunol 102, 1309-1313.-   Benjamin R J, Cobbold S P. Clark M R and Waldmann H. 1986. Tolerance    to rat monoclonal antibodies: implications for serotherapy. J Exp    Med 163, 1539-1552.-   Bird R E, Hardman K D, Joacobson J W. 1988. Single-chain    antigen-binding proteins. Science 242, 423-426.-   Brown J H, Jardetzky T S, Gorga J C, Stern″, Urban R G, Strominger J    L, Wiley D C. 1993. Three-dimensional structure of the human class    II histocompatability antigen HLA-DR1. Nature 364, 33-39.-   Chiller J M, Habicht G S, Weigle W O. 1971. Kinetic differences in    unresponsiveness of thymus and bone marrow cells. Science 171,    813-815.-   Chothia C and Lesk A M 1987. Canonical structures for the    hypervariable regions of immunoglobulins. J Mol. Biol. 196, 901-917.-   Chothia C, Lesk A M, Tramontano A, Levitt M, Smith-Gill S J. Air G,    Sheriff S, Padlan E A, Davies D, Tulip W R, Colman P M, Spinelli S,    Alzari P M, Poljak R J. 1989. Conformations of immunoglobulin    hypervariable regions. Nature 342, 877-883.-   Chothia C, Lesk A M, Gherardi E, Tomlinson I M, Walter G, Marks J D,    Llewelyn, M 8, Winter G. 1992. Structural repertoire of the human VH    segments. J Mol Biol 227, 799-817.-   Cobbold S P. Clark M R. Benjamin R J, Waldmann H. Monoclonal    antibodies and their use. EP 0 536 807 and EP 0 240 344. Wellcome    Foundation Ltd.-   Cunningham, B. C. and Wells, J. A. 1989. High resolution epitope    mapping of hGH-receptor interactions by alanine-scanning    mutagenesis. Science, 244, 1081-1085.-   Gilliland L K, Norris N A, Marquardt H, Tsu T T. Hayden M S,    Neubauer M G, Yelton D E. Mittler R S, Ledbetter J A. 1996. Rapid    and reliable cloning of antibody variable regions and generation of    recombinant single chain antibody fragments. Tissue Antigens, 47,    1-20.-   Gorman S D, Clark M R, Routledge, E G, Cobbold S P,    Waldmann H. 1991. Reshaping a therapeutic CD4 antibody. Proc Natl    Acad Sci USA 88, 4181-4185.-   Gram H, Marconi L A, Barbas C F 3rd, Collet T A, Lerner R A, Kang    A S. 1992. In vitro selection and affinity maturation of antibodies    from a naive combinatorial immunoglobulin library. Proc Natl Acad    Sci USA 89, 3576-3580.-   Greaves D R. Wilson F D, Lang G, Kioussis D. 1989. Human CD2    3′-flanking sequences confer high-level, T-cell specific,    position-independent gene expression in transgenic mice. Cell 56,    979-986.-   Hale G, Xia M-Q, Tighe H P, Dyer M J S, Waldmann H. 1990. The    CAMPATH-1 antigen (CDw52). Tissue Antigens 35, 118-127.-   Hale G, Hoang T, Prospero T, Watt S M, Waldmann H. 1983. Removal of    T cells from bone marrow for transplantation: comparison of rat    mono-clonal anti-lymphocyte antibodies of different isotypes. Mol    Biol Med 1, 305-319.-   Huston J S, Mudgett-Hunter M, Tai M-S, McCartney J, Warren F. Haber    E, Oppermann H. 1991. Protein engineering of single-chain Fv analogs    and fusion proteins. Methods Enzymol 203, 46-88.-   Hoogenboom H R, Griffiths A D, Johnson K S, Chiswell D J, Hudson P,    Winter G. 1991. Multi-subunit proteins on the surface of filamentous    phage: methodologies for displaying antibody (Fab) heavy and light    chains. Nucl Acids Res 19, 4133-4137.-   Hunt D F, Michel H, Dickinson T A, Shabanowitz J, Cox A L, Sakaguchi    K, Apella E, Grey H M, Sette A. 1992. Peptides presented to the    immune system by the murine class II major histocompatability    molecule I-Ad. Science 256, 1817-1820.-   Huston J S, Levinson D, Mudgett-Hunter M et al. 1988. Protein    engineering of antibody binding sites: recovery of specific activity    in an anti-digoxin single-chain Fv analogue produced in Escherichia    coli. Proc Natl Acad Sci USA, 85, 5879-5883.-   Isaacs J D, Watts R A, Hazleman B L et al. 1992. Humanized    monoclonal antibody therapy for rheumatoid arthritis. Lancet 340,    748-752.-   Isaacs J D, Waldmann H. 1994. Helplessness as a strategy for    avoiding antiglobulin responses to therapeutic monoclonal    antibodies. Therapeutic Immunol 1, 303-312.-   Jones P T, Dear P H, Foote J, Neuberger M S, Winter G. 1986.    Replacing the complementarity-determining regions in a human    antibody with those from a mouse. Nature 321, 522-525.-   Kabat E A, Wu T T, Reid-Miller M, Perry H M, Gottesman K S. 1987.    Sequences of Proteins of Immunological Interest, 4th ed. Washington    DC, Public Health Service, NIH.-   Levitskaya J, Coram M, Levitsky V, Imreh S, Steigerwald-Mullen P M,    Klein G, Kurilla M G, Masucci M G. 1995. Inhibition of antigen    processing by the internal repeat region of the Epstein-Barr virus    nuclear antigen-1. Nature 375, 685-488.-   Lockwood C M, Thiru S, Isaacs J D, Hale G, Waldmann H. 1993.    Long-term remission of intractable systemic vasculitis with    monoclonal antibody therapy. Lancet 341, 1620-1622.-   Low N M, Hollinger P H, Winter G. 1996. Mimicking somatic    hypermutation: affinity maturation of antibodies displayed on    bacteriophage using a bacterial mutator strain. J Mol Biol 260,    359-368.-   Miranda J J Zola H, Howard J G. 1973. Studies on immunological    paralysis. X. Cellular characteristics of the induction and loss of    tolerance to leva (polyfructose). Immunology 23, 843-855.-   Novotny J, Bruccoleri R E, Newell J, Murphy D, Haber E,    Karplus M. 1983. Molecular anatomy of the antibody binding site. J    Biol Chem 258, 14433-14437.-   Page M J. Sydenham M A. 1991. High level expression of the humanized    monoclonal antibody Campath-1H in Chinese hamster ovary cells.    Biotechnol. 9, 64-68.-   Parish C R, Ada G L. 1969. The tolerance inducing properties in rats    of bacterial flagellin cleaved at the methionine residues.    Immunology 17, 153-164.-   Riechmann L, Clark M, Waldmann H, H, Winter G. 1988. Reshaping human    antibodies for therapy. Nature 332, 323-327.-   Routedge E G, Gorman S D, Clark M R. 1993. Reshaping antibodies for    therapy. In Protein Engineering of Antibody Molecules for    Prophylactic and Therapeutic Applications in Man. ed. M Clark,    Academic Titles, Nottingham, UK. pp 13-44.-   Routledge E G, Lloyd I, Gorman S D, Clark M, Waldmann H. 1991. A    humanized monovalent CD3 antibody which can activate homologous    complement. Eur J Immunol 21, 2717-2725.-   Rudensky A Y, Preston-Hurlburt P, Hong S, Barlow A, Janeway C A    Jr. 1991. Sequence analysis of peptides bound to MHC class II    molecules. Nature 353, 622-627.-   Rudensky A, Janeway C A Jr. 1993. Studies on Naturally processed    peptides associated with MHC class II molecules. In Sette A (ed.)    Naturally Processed Peptides. Basel, Karger, pp 134-151.-   Xia M-Q Tone M, Packman L. Hale G, Waldmann H. 1991.    Characterization of the CAMPATH-1 (CDw52) antigen: biochemical    analysis and cDNA cloning reveal an unusually small peptide    backbone. Eur J Immunol 21, 1677-1684.

1-23. (canceled)
 24. A method for inducing immunological tolerance in ahost to a therapeutic antibody having an affinity for a cell-surfaceantigen, comprising: administering a non-immunogenic non-cell bindingantibody or a fragment thereof to a host to tolerize the host to thetherapeutic antibody, wherein the non-immunogenic non-cell bindingantibody is produced by identifying one or more amino acid residues inthe complementary determining region (CDR) of the therapeutic antibodywhich are involved in antigen binding, and modifying one or more of theidentified amino acid residues in the CDR of the therapeutic antibody toproduce a non-immunogenic variant antibody which retains epitopesincluding idiotypic determinants of the original therapeutic antibodyand which is a non-cell binding antibody, wherein the non-immunogenicnon-cell binding antibody is a single antibody which (1) has affinityfor antigen binding reduced to 50% or less as compared to thetherapeutic antibody due to the modification(s), (2) comprises at leastone epitope present in the therapeutic antibody which induces an immuneresponse, (3) induces immunological tolerance to the therapeuticantibody, and (4) has variable domains with greater than 90% sequenceidentity with the variable domains of the therapeutic antibody, andwherein the non-immunogenic non-cell binding antibody is not a mixedmolecule antibody having an H or L chain of a therapeutic antibodypaired with an L or H chain of an unrelated antibody, wherein thefragment of the non-immunogenic non-cell binding antibody is produced byfragmenting the non-immunogenic non-cell binding antibody producedabove, wherein the fragment (1) comprises the modification(s) of thenon-immunogenic non-cell binding antibody, (2) has reduced affinity forantigen binding as compared to the therapeutic antibody due to themodification(s), (3) comprises at least one epitope present in thetherapeutic antibody which induces an immune response, and (4) inducesimmunological tolerance to the therapeutic antibody.
 25. The methodaccording to claim 24, wherein the therapeutic antibody is administeredafter the administration of the non-immunogenic non-cell bindingantibody or fragment thereof.
 26. The method according to claim 24,wherein the amino acid residue(s) is/are identified using one or moretechniques selected from the group consisting of X-ray crystallography,computer modeling, comparative modeling, CDR swapping, Alanine scanning,phage display and cycling of V-region genes.
 27. The method according toclaim 24, wherein the amino acid residue(s) is/are modified by geneticmanipulation.
 28. The method according to claim 27, wherein the geneticmanipulation is site-directed mutagenesis.
 29. The method according toclaim 24, wherein the affinity for antigen binding of thenon-immunogenic non-cell binding antibody is reduced to 10% or less ascompared to the therapeutic antibody.
 30. The method according to claim29, wherein the affinity for antigen binding of the non-immunogenicnon-cell binding antibody is reduced to 1% or less as compared to thetherapeutic antibody.
 31. The method according to claim 24, wherein thenon-immunogenic non-cell binding antibody has greater than 90% aminoacid sequence identity with the therapeutic antibody.
 32. The methodaccording to claim 31, wherein the non-immunogenic non-cell bindingantibody has greater than 95% amino acid sequence identity with thetherapeutic antibody.
 33. The method according to claim 32, wherein thenon-immunogenic non-cell binding antibody has greater than 99% aminoacid sequence identity with the therapeutic antibody.
 34. The methodaccording to claim 24, wherein the non-immunogenic non-cell bindingantibody comprises variable domains having greater than 95% amino acidsequence identity with variable domains of the therapeutic antibody. 35.The method according to claim 34, wherein the non-immunogenic non-cellbinding antibody comprises variable domains having greater than 99%amino acid sequence identity with variable domains of the therapeuticantibody.
 36. The method according to claim 24, wherein thenon-immunogenic non-cell binding antibody comprises constant domainshaving greater than 90% amino acid sequence identity with constantdomains of the therapeutic antibody.
 37. The method according to claim36, wherein the non-immunogenic non-cell binding antibody comprisesconstant domains having greater than 95% amino acid sequence identitywith constant domains of the therapeutic antibody.
 38. The methodaccording to claim 37, wherein the non-immunogenic non-cell bindingantibody comprises constant domains having greater than 99% amino acidsequence identity with constant domains of the therapeutic antibody. 39.The method according to claim 38, wherein the non-immunogenic non-cellbinding antibody comprises constant domains identical with constantdomains of the therapeutic antibody.
 40. The method according to claim24, wherein the non-immunogenic non-cell binding antibody comprisesframework regions having greater than 90% amino acid sequence identitywith framework regions of the therapeutic antibody.
 41. The methodaccording to claim 40, wherein the non-immunogenic non-cell bindingantibody comprises framework regions having greater than 95% amino acidsequence identity with framework regions of the therapeutic antibody.42. The method according to claim 41, wherein the non-immunogenicnon-cell binding antibody comprises framework regions having greaterthan 99% amino acid sequence identity with framework regions of thetherapeutic antibody.
 43. The method according to claim 24, wherein themodification in the CDR of the therapeutic antibody is located in VHCDR2.
 44. The method according to claim 24, wherein the modificationcomprises a single or a double amino acid substitution.
 45. The methodaccording to claim 44, wherein the single or double amino acidsubstitution is located in VH CDR2.
 46. The method according to claim24, wherein the therapeutic antibody has affinity for CD52.
 47. Themethod according to claim 46, wherein the therapeutic antibody is ahumanized Campath-1 antibody.
 48. The method according to claim 24,wherein the non-immunogenic non-cell binding antibody comprises foreignCDRs with respect to the constant region of the non-immunogenic non-cellbinding antibody.
 49. The method according to claim 24, wherein thenon-immunogenic non-cell binding antibody comprises foreign CDRs withrespect to the heavy and light chain variable domain framework regionsof the non-immunogenic non-cell binding antibody.
 50. The methodaccording to claim 24, wherein the non-immunogenic non-cell bindingantibody comprises non-CDR regions of human origin.
 51. The methodaccording to claim 24, wherein each of the modified amino acidresidue(s) reduces the affinity for antigen binding of thenon-immunogenic non-cell binding antibody.
 52. The method according toclaim 24, wherein the affinity for antigen binding of thenon-immunogenic non-cell binding antibody is reduced such that theantibody does not show detectable binding to antigen in ELISAs at 100times the minium concentration at which binding of the therapeuticantibody is detectable.
 53. The method according to claim 52, whereinthe non-immunogenic non-cell binding antibody does not show detectablebinding to antigen in ELISAs at 1,000 times the minium concentration atwhich binding of the therapeutic antibody is detectable.
 54. The methodaccording to claim 53, wherein the non-immunogenic non-cell bindingantibody does not show detectable binding to antigen in ELISAs at 10,000times the minium concentration at which binding of the therapeuticantibody is detectable.
 55. The method according to claim 24, whereinthe fragment is a monovalent or divalent fragment of the non-immunogenicnon-cell binding antibody.
 56. The method according to claim 55, whereinthe monovalent or divalent fragment of the non-immunogenic non-cellbinding antibody is Fab, Fab′ or F(ab′)₂.
 57. The method according toclaim 55, wherein the fragment is a single chain antibody.